Structure activity relationship analysis of antiproliferative cyclic C5-curcuminoids without DNA binding: Design, synthesis, lipophilicity and biological activity

Article abstract of DOI:10.1016/j.molstruc.2019.127661

The chemical susceptibility of the β-diketone linker between the two aromatic rings in the structure of curcumin to hydrolysis and metabolism has made it crucial to investigate structurally modified analogs of curcumin without such shortcomings. The synth

Full text of DOI:10.1016/j.molstruc.2019.127661

Journal Pre-proof
Structure activity relationship analysis of antiproliferative cyclic C -curcuminoids
5
without DNA binding: Design, synthesis, lipophilicity and biological activity
Imre Huber, Zsuzsanna Rozmer, Zoltán Gyöngyi, Ferenc Budán, Péter Horváth,
Eszter Kiss, Pál Perjési
PII:
S0022-2860(19)31770-3
DOI:
https://doi.org/10.1016/j.molstruc.2019.127661
MOLSTR 127661
Reference:
To appear in: Journal of Molecular Structure
Received Date: 31 October 2019
Accepted Date: 26 December 2019
Please cite this article as: I. Huber, Z. Rozmer, Zoltá. Gyöngyi, F. Budán, Pé. Horváth, E. Kiss, Pá.
Perjési, Structure activity relationship analysis of antiproliferative cyclic C -curcuminoids without DNA
5
binding: Design, synthesis, lipophilicity and biological activity, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2019.127661.
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O
O
CHO
+
2
X
R
R
X
R
5
1-4
6-9
O
O
O
O
CHO
N
H
2
N
R
CH3
CH3
3
OH
5a-e
1
4
R: a = NO2; b = Cl; c = H; d = OCH3; e = N(CH3)2

Structure activity relationship analysis of antiproliferative
cyclic C5-curcuminoids without DNA binding: design,
synthesis, lipophilicity and biological activity
Imre Hubera,*, Zsuzsanna Rozmera, Zoltán Gyöngyib, Ferenc Budánb, Péter Horváthc,
Eszter Kissc, Pál Perjésia
a Department of Pharmaceutical Chemistry, University of Pécs, Pécs H-7624,
Hungary
b Department of Public Health Medicine, Medical School, University of Pécs, Pécs H-
7624, Hungary
c Department of Pharmaceutical Chemistry, Semmelweis University, Budapest H-
1092, Hungary
Abstract
The chemical susceptibility of the ß-diketone linker between the two aromatic rings in the structure of
curcumin to hydrolysis and metabolism has made it crucial to investigate structurally modified analogs
of curcumin without such shortcomings. The synthesis of twenty cyclic C5-curcuminoids is described in
this study in order to gain more insight into their anticancer structure-activity relationship (SAR). The
design of their synthesis included four different cyclanones and five substituted aromatic aldehydes to
form four, five-membered subgroups. These model compounds were evaluated in vitro for
antiproliferative activity in an XTT cell viability assay against MCF-7 human non-invasive breast
adenocarcinoma cancer cells and Jurkat human T lymphocyte leukemia cells in five different
concentrations (10 nM, 100 nM, 1 ꢀM, 10 ꢀM and 20 ꢀM). The majority of the compounds investigated
have shown remarkable cytotoxicity with IC50 values in the range of 120 nM and 2 ꢀM with very high
relative toxicity values to curcumin. The SAR conclusions are drawn and summarized. A method was
developed and applied in a TLC based experimental logP measurement, which is new for such C5-
curcuminoids. The logP data and structural modifications have shown a strong correlation. The
correlation of these experimental logP and the corresponding IC50 values of the model-compounds
were calculated according to the Pearson and Kendall correlation coefficient and showed weak
concordance. The physicochemical behaviors of the majority of these compounds are in good
accordance with Lipinski’s rule. The most promising compound is 7a, which is the most active (IC50
=
0.12-0.32 ꢀM), most potent (80 times of curcumin) with the lowest lipophilicity (experimental logP =
3.22) which is important also from a pharmacokinetic point of view. The analysis of experimental logP

and computed ClogP values have revealed good agreement. These cyclic C5-curcuminoids in contrast
to curcumin do not bind to natural DNA based on their CD spectra.
Key words: cyclic C5-curcuminoids; cytotoxic; experimental logP; SAR; DNA binding; antiproliferative
1. Introduction
Plants are unbeatable sources of nutraceuticals. They are also considered to be one of the major
sources of lead molecules and drug candidates [1]. The intense yellow rhizome of herbs in the
Zingiberaceae (ginger) family provides turmeric. Species like Curcuma longa L. and/or Curcuma
domestica L. for example, are used to prepare that well-known spice nowadays. The major component
of turmeric is its secondary metabolite, curcumin (Fig 1). The preventive and therapeutic applications
of curcumin are extremely diverse. Many clinical trials have been conducted to evaluate their
pharmacokinetics, safety and efficacy. In cancer, cardiovascular diseases, diabetes, inflammatory
diseases and communicable diseases, chronic arsenic exposure and alcohol intoxication, curcumin
has been analyzed [2].
Extensive research has shown that curcumin exhibits many different pharmacological effects and has
a number of molecular targets with multiple pathways as an antiproliferative molecule [3, 4]. Curcumin
(diferuloylmethane), 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadien-3,5-dion has a ß-diendione
linkage (1,6-heptadien-3,5-dion) containing seven carbon atoms (C7) between the two arylidene
groups in its structure (Fig. 1). It was a belief for a long time, that natural curcuminoids (including the
metabolites of curcumin) are exclusively C7-curcuminoids. However, along with curcumin, a C5-
curcuminoid was also isolated from both Curcuma longa and Curcuma domestica [5, 6]. This natural
C5-curcumin [7], 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one, as a truncated analog of
curcumin contains a C5 ß-dienone linker (1,4-pentadiene-3-one) between the two benzylidene cores in
its structure (Fig. 1).
Fig. 1. Natural curcumins and synthetic homocyclic or heterocyclic C5-curcuminoids

This compound, C5-curcumin and its related derivatives proved to be more potent anticancer
molecules compared to curcumin [7, 8]. Their interesting reversible thia-Michael reaction, which is
principal in binding to the biological place of action of such curcuminoids is described in detail [8].
In view of the discovery of C5-curcumin showing superior anticancer activity to curcumin, a number of
new cyclic C5-curcuminoids have been synthesized. Structural modifications of C5-curcuminoids
focusing on enhancing their bioactivities have been investigated intensively during the last couple of
decades. Curcumin, C5-curcumin and cyclic C5-curcuminoids were subjected to structure-activity
relationship (SAR) studies [3, 6-9] in order to find details on the most appropriate structural changes
for the best cytotoxic effect.
The synthetic, cyclic C5-curcuminoid with a cyclohexanone core (Fig. 1) for example proved to be
more active against castration-resistant prostate cancer compared to curcumin both in vitro and in vivo
[9]. Numerous studies have been conducted also on the (3E,5E)-3,5-dibenzylidene-4-piperidone
synthetic, heterocyclic C5-curcuminoid family [10-15]. It became clear about these 4-piperidone
derivatives that they exhibit higher cytotoxicity than curcumin towards different tumor cell lines like
breast, prostate, cervix, melanoma, etc. [3]. Compound EF31 for example (Fig. 1), similarly to related
derivatives is shown to be a pleiotropic inhibitor of kinases (relevant to many forms of cancer), that
operate at multiple points along cell signaling pathways. In addition to superior cytotoxicity to
curcumin, these 4-piperidone derivatives show differential cytotoxicity; they are less toxic to non-
cancer cells when compared to cancer cells [10, 13, 16, 17].
Another crucial advantage of these synthetic C5-curcuminoids is that a number of them are able to
revert multi-drug resistance (MDR) [14, 16, 17]. Moreover, there are reports on the in vivo tolerability
and lack of acute toxicity of these curcuminoids on rodents [18]. A number of C5-curcuminoids have
several different modes of action, such as inducing apoptosis, cell cycle arrest, inhibiting the
biosynthesis of polypeptides fundamental to tumor-progression, affecting mitochondrial respiration and
stimulation/inhibition of certain enzymes playing a role in tumor growth [19].
These cyclic C5-curcuminoids bear a ß-dienon cross-conjugated moiety essential for cytotoxicity [22]
in their structure. This ß-dienon function is a reactive and selective Michael-acceptor [8]. Selectivity
appears in the fact that the Michael-reaction takes place exclusively with the nucleophilic thiol groups
of biological macromolecules. It seems to be logical, that these curcuminoids cannot react with DNA in
contrast to curcumin [20, 21]. To gain details and proof of their mode of action in this respect we
decided to perform CD spectroscopic investigations. Such CD spectroscopic data about the cyclic and
heterocyclic C5-curcuminoids are not available in the corresponding literature to our knowledge. In
view of the considerations above as a continuation of our previous SAR studies [22, 23], and to gain
more insight into the influence of structural modifications on the cytotoxic activity of such homocyclic
and heterocyclic C5-curcuminoids we resynthesized twenty previously described molecules (see
experimental part) for a systematic SAR study. These C5-curcuminoids are less susceptible to
metabolism compared to curcumin [24, 25]. We selected the following two cell-lines to perform XTT
cell viability assays: MCF-7 (human non-invasive breast cancer cells) and Jurkat (human T
lymphocyte leukemia cells). We applied an RP-TLC method to measure the experimental logP values

of the twenty selected molecules. Some physicochemical parameters of the molecules were also
computed in order to obtain the corresponding calculated ClogP values for example.
2. Experimental
2.1. Chemical synthesis
For the synthesis of the model compounds of this study, we have used a previously described “one
pot” method of ours [22]. These compounds are known from the chemical literature and were prepared
according to known methods, like 6a [38], 6b, 6c, 6d [39], 6e [40], 7a, 7d, 7e [41], 8a, 8d [42] and 8e
1
[43]. We have recorded their H-NMR spectra and measured their melting points. Our own physical
data are in good accordance with the published parameters. Chemicals, solvents and reagents for the
study were purchased from Alfa Aesar, Molar and Merck Ltd (Budapest, Hungary). Melting points were
determined on a Barnstead-Electrothermal 9100 apparatus and are uncorrected. Silica gel 60 (0.2-0.5
mm, MERCK) was used for column chromatography and pre-coated silica gel 60 (F-254, MERCK)
plates for TLC.
1
NMR: H-NMR spectra were obtained using a Varian UNITY INOVA 400 WB spectrometer. Chemical
shifts are referenced to the residual solvent signal. Measurements were run at a probe temperature of
1
298K in CDCl3 or DMSO-d6 solutions. All the H-NMR spectra were in accordance with the expected
structures.
2.2. CD: CD and UV measurements were performed on a Jasco J-720 spectropolarimeter (Jasco LtD.,
Tokyo, Japan) in Jasco cylindrical cuvettes with a path length of 10 mm. 1 mg of chicken erythrocyte
DNA (Reanal, Budapest, Hungary) was dissolved in 10 ml distilled water (stock solution, 0.1 mg/ml)
and was further diluted to 0.033mg/ml for the experiments. Tested substances were dissolved in
DMSO (Sigma-Aldrich, Budapest, Hungary) in 5-10 mM concentration. All experiments were
performed at ambient temperature.
2.3. Physicochemical calculations:
For the prediction of some physicochemical parameters of our compounds, we have used the
Calculator Plugins of ChemAxon. The ClogP values were calculated with all three methods and
database of the software. The ClogP and solubility values are results in physiological pH=7.4. See the
corresponding homepage for detailed conditions of the calculations [33]. To explain the relationship
between biodata and calculated physicochemical parameters the Lipinski approaches have been used
[32].
2.4. Cell culture
MCF-7 human non-invasive breast adenocarcinoma cancer cells [26] were cultured in DMEM medium
with 4.5 g/l glucose and 2 mM L-glutamine (Lonza, Verviers, Belgium). Jurkat human T lymphocyte
leukemia cells [27] were cultured in RPMI 1640 medium with 2 mM L-glutamine (Lonza). All media

were supplemented with 10 % heat-inactivated FBS (Sigma, St. Louis, MO) and antibiotics 10 ml/l
(penicillin-streptomycin 100 x, Sigma, St. Louis, MO).
2.5. Cytotoxicity test
Following the protocol of XTT cell viability assay (Biotium, Hayward, CA), the concentrations were
50.000 cells/ml (MCF-7) and 100.000 cells/ml (Jurkat) in the above described supplemented media.
100 µl of cell suspension was placed into each well of 96-well plates and incubated for 48 hours in
regular conditions (37 °C, 100 % relative humidity and 5 % CO2). 5 µl solutions of curcumin and its
analogs diluted in DMSO (Sigma, St. Louis, MO), added to each well to reach 10 nM, 100nM, 1 µM,
10 µM and 20 µM final concentrations. After 24-hour incubation in regular cell culture conditions, 25 µl
activated XTT solution was mixed with media in the wells and consecutively incubated for 3 hours.
Finally, the absorbance was measured at a wavelength of 490 nm with DiaReader ELx800 (Dialab,
Vienna, Austria) microplate reader. Results of curcumin and its analogs were compared to the DMSO
controls with Student’s t-test and statistical significance (p<0.05) was calculated.
2.6. Experimental (logP):
RP-TLC logP determinations were performed by a slight modification of the previously optimized RP-
TLC method used for determination of some chalcones and cyclic chalcone analogs [28, 29].
Compounds of the calibration set (selected chalcones and cyclic chalcone derivatives, see the
structures in “Supportive material”) were synthesized and purified as described [37]. Their logP values
were determined earlier [28, 29]. Two compounds of the validation set (progesterone, diazepam) were
of pharmacopoeial grade. All other reagents used were of analytical grade.
RP-TLC determination of the logP of the cyclic C5-curcuminoid compounds 6-9 was performed
on 20cm x 20cm plates precoated with 0.25 mm layers of silanized silica gel 60F254 (Merck, Germany;
#5747). The plates were washed with methanol and dried before use. The samples of 6-9 were
dissolved in 1:1 methanol-chloroform (c = 1 mg/cm3) and 2 ꢀl of these solutions were spotted on the
plate. Methanol-water, 70 + 30 (v/v), was used as mobile phase. The paper-lined chromatographic
chamber was saturated with the mobile phase for 30 min before use. After development (150 mm) the
plates were dried and the chromatograms assessed visually under UV illumination (λ = 254 nm).
Three TLC determinations were performed for each substance.
Some of the investigated molecules contain weakly basic functional groups. It was found,
however, that they occur as non-ionized, neutral species under the experimental conditions.
2.7. Validation of the RP-TLC system:
The optimized chromatographic system underwent validation prior to logP measurements. For
this purpose, four molecules (progesterone, diazepam, chalcone and a chalcone derivative (Q-693))
with known logP values were tested. Comparison of their logPTLC values obtained in this work with
previously published experimental logP data [28] resulted in rather good agreement. Thus, these four
compounds were also added to the calibration set.

Table 6 LogPSF and logPTLC values of the compounds of the validation set
logPSF
3.62
3.74
3.54
2.82
logPTLC
3.62
∆logP
0
Chalcone
Q693
3.77
0.03
0.13
0.23
Progesterone
Diazepam
3.67
3.05
The logPTLC data obtained by our optimized and validated RP-TLC method are listed in Table 6. The
logP data can provide a good basis for evaluation of structure-lipophilicity relationship for the
examined compounds 6-9. See “Supplementary data” for more details.
3. Results and discussion
3.1. Chemistry
The synthesis of our model compounds 6-9 for this SAR study was performed according to known
methods involving the conditions of a Claisen-Schmidt condensation as depicted in Fig. 2. The
nitrogen containing heterocyclic derivatives 7 and 8 have been prepared according to our new “one-
pot” synthesis [22] starting from the corresponding ß-ketoesters and aldehydes. Condensation
reactions were carried out under basic or acidic conditions in good yields in a range of 75-90%.
Formally, four different cyclic ketones, like 4-methylcyclohexanone (1), 4-piperidone (2), N-methyl-4-
piperidone (3) or 4-hydroxycyclohexanone (4) and always the same five benzaldehydes (5a-e) have
been transformed in these cross-aldol condensation reactions to form the desired model compounds
6-9. Cyclanone 4 was prepared accordingly to our previously published method [23].

Fig. 2. Synthetic homo- and heterocyclic C5-curcuminoid model compounds 6-9 with four different
cyclanone cores and five aromatic substituents
Our goal was on the one hand to prepare target compounds 6-9 with a small substituent in the central
ring (X=CH-CH3, N-H, N-CH3 or CH-OH) due to the fact, that derivatives 7b and 7c (Fig.3) turned out
to be very promising antiproliferative agents. They have even been selected as standard lead
compounds to be compared to newer promising agents [22 and references therein]. We selected five
substituents onto the aromatic rings (nitro, chloro, hydrogen, methoxy, dimethylamino) on the other
hand, in order to find the most optimal structure from the SAR analysis with the two substituents in the
central and aromatic rings. In Fig. 3 we can see the structures of the twenty cyclic C5-curcuminoid
derivatives prepared for this study. Compounds 7b, 7c, 8b, 8c [22] and 9a-e [23] were prepared by us
previously together with the others (6a-e, 7a, 7d, 7e, 8a, 8d and 8e, see the experimental part) are in
good accordance with the literature data in terms of their melting points and 1H NMR spectra.
Fig. 3. Structures of the twenty homo- and heterocyclic C5-curcuminoids prepared for this SAR study
3.2. Cytotoxicity
The synthesized compounds were evaluated for their in vitro antiproliferative activity against MCF-7
human non-invasive breast adenocarcinoma cancer cells [26] and Jurkat human T lymphocyte
leukemia cells [27] in an XTT cell viability assay. Inhibition of cell proliferation by these active
compounds at various concentrations (10 nM, 100 nM, 1 ꢀM, 10 ꢀM and 20 ꢀM) was measured, and
their IC50 (the concentration that causes a 50% cell proliferation inhibition) values were calculated and
are summarized in Table 1.

It was observed generally, that Jurkat leukemia cells were more susceptible to cytotoxic treatment in
almost all cases compared to MCF-7 cells (Table 1). We have observed in eight cases, that the given
compound was practically ineffective. Compounds 6e and 8e were ineffective on both cell lines.
Although we did not use non-cancer cells for cytotoxicity tests, the difference in susceptibility of the
two cell lines and of ineffective cases of these compounds is proof of their selective toxicity. We can
state, however, that the majority of these compounds proved very good activity on cytotoxicity/viability
tests. Many of them like 6a, 7a, 7b, 7c, 7e, 8a, 8b and 9a (6a, 7a, 7c and 8b on both cell lines) were
active even in the nanomolar range.
Table 1 In vitro antiproliferative activities of model-compounds against Jurkat and MCF-7 cancer cell
lines in terms of their IC50 (the concentration that causes a 50% cell proliferation inhibition) values in
micromoles and relative potentials related to Curcumin
Compound
Jurkat
MCF-7
Relative pot.a
Relative pot.a
IC50 [µM]
IC50 [µM]
0.95
0.40
6,59
4.00
2.89
>20
1.00
2.38
9.76
0.49
17.00
9.80
8.77
>20
1.00
19.92
Curcumin
6a
0.14
0.57
6b
0.24
0.996
6c
0.33
1.32
6d
not relevant
2.97
not relevant
81.33
6e
0.32
0.36
0.24
10.32
0.14
1.00
0.21
1.49
9.32
>20
0.12
>20
7a
2.64
not relevant
12.34
7b
3.96
0.81
5.87
9.80
0.75
0.43
2.38
>20
7c
0.09
1.66
7d
6.79
0.996
7e
0.95
13.32
8a
4.52
22.70
8b
0.64
4.31
8c
0.10
not relevant
not relevant
5.95
8d
not relevant
1.37
>20
8e
0.88
1.99
2.30
10.50
19.85
1.64
>20
9a
0.48
not relevant
1.32
9b
0.41
8.71
11.31
>20
9c
0.09
0.86
9d
0.05
not relevant
9e
a
Relative potential: IC50 value of curcumin divided by the IC50 value of the compound.
Moreover, many of them showed superior cytotoxic activity to Curcumin, namely compounds 7e, 8b,
7c, 7a, 7b and 6a on Jurkat cells while compounds 7a, 8b, 6a, 8a and 7c on MCF-7 cell line. The
previous sequence is important because it shows also the ranking of their IC50 values. The IC50 values

of the most active compounds on Jurkat cells are between 0.14 (7e) and 0.40 (6a) ꢀM while 0.12 (7a)
and 0.81 (7c) ꢀM on MCF-7 cells.
The relative potential, which shows the rate of dominance over curcumin of compounds in Table 1, is
higher on MCF-7 cell lines in general. For example, 7e on Jurkat and 7a on MCF-7 cells have a similar
IC50 value (0.14 and 0.12 respectively), but the relative potential is different. It is 6.79 for 7e on Jurkat
and 81.33 for 7a on MCF-7. This means, that 7a is the most potent congener compared to curcumin. It
is important to emphasize, that derivatives 6a, 7a, 7c and 8b possess cytotoxicity on both cell lines.
We can draw a very positive conclusion that our model compounds are showing selective toxicity with
different relative potentials.
Fig. 4. The primary (A) and secondary (B) pharmacophore functions of cyclic C5-curcuminoids
The ß-dienone moiety, which serves as the primary pharmacophore function (Fig. 4, A) in the structure
of our compounds 6-9 is the same in all derivatives. In order to compare changes in activity after
structural modifications on the central ring and on the aromatic benzylidene rings we have created a
“heat-map” including the structures of the compounds with substituents on the central and aromatic
rings, IC50 values of our cyclic C5-curcuminoids and the two cell lines (Table 2). The smaller IC50
values (higher antiproliferative activity) are in red whilst the bigger IC50 values (lower activity) are in
blue. The others in between are in yellow and green.
Table 2 The IC50 “heat-map” of compounds 6-9. Curcumin IC50: Jurkat – 0.95; MCF-7 – 9.76 ꢀM

R=
Compound
nitro
a
chloro
b
H
methoxy dimethylamino
Cell line:
Jurkat
c
d
e
0.40
0.49
0.32
0.12
1.00
0.75
0.88
1.64
0.70
6.59
17.00
0.36
>20
4.00
9.80
0.24
0.81
1.49
2.38
2.30
8.71
3.72
2.89
8.77
10.32
5.87
9.32
>20
>20
>20
0.14
9.80
>20
>20
19.85
>20
9.93
6
(CH-CH3)
7
MCF-7
Jurkat
(N-H)
8
MCF-7
Jurkat
0.21
0.43
1.99
>20
(N-CH3)
9
MCF-7
Jurkat
10.50
11.31
8.43
(CH-OH)
MCF-7
Average:
4.43
Our first conclusion is that the heterocyclic derivatives 7 and 8 exhibited more pronounced
antiproliferative activities compared to their homocyclic counterparts 6 and 9. This fact promotes our
earlier findings [22, 23] on such heterocyclic C5-curcuminoids including compounds 7b, 7c, 8b and 8c
[22] or 9a-e [23]. The reference standard was cisplatin in both of these two studies. The position
opposite to the ketone carbonyl on the central cyclanone ring, being an auxiliary binding ability
(secondary pharmacophore, Fig. 4, B) to the in vivo biological site of action, has a great influence on
the cytotoxicity of these compounds. In other words, the binding strength of these cyclic C5-
curcuminoids to the biological site of action is stronger when there is a suitable substituent and/or a
nitrogen heteroatom at this position. This moiety on the central cyclanone ring (Fig. 4, B) is able to
enhance the interaction between the cyclic C5-curcuminoids and their biological place of action in the
cancer cell. This is visible on the example of 6b and 8b: the cytotoxic activity increases on both cell
lines (see the corresponding IC50 values) after the nitrogen atom “appears” in the homocycle of 6b to
form the heterocyclic 8b counterpart (Table 2). A similar example is the exchange of the NH functional
group of compound 7c to an OH in 9c.
The second conclusion is that the electron withdrawing p-chloro and especially p-nitro substituents on
the benzylidene parts are dominating the p-methoxy or p-dimethylamino electron donor substituents.
For example, compound 6a with a nitro group showed 0.40 ꢀM IC50 value on Jurkat cells, whilst 6e
with a dimethylamino substituent was practically ineffective. The same is true on the MCF-7 cell line
about 6a and 6e. Apart from some exceptions, similar examples are there in Table 2. A definite
structure/activity relation was discovered comparing the average IC50 values of the five substituents
(nitro, chloro, hydrogen, methoxy and dimethylamino) on benzylidene moieties (Table 2). The best
performer is the nitro-substituted benzylidene group (6a, 7a, 8a and 9a) with IC50 values ranging from
0.12 and 1.64 ꢀM. The second in this sequence is the unsubstituted benzylidene group (6c, 7c, 8c
and 9c), the third is the chloro-substituted 6b, 7b, 8b and 9b. The last two positions remain to the
methoxy (d) and dimethylamino (e) substituents with average IC50 values of 8.43 and 9.93 ꢀM
respectively.

Experimental and calculated data in Table 1 and 2, in relation to the two moieties A and B (Fig. 4)
suggest that there is an electron withdrawing substituent on the benzylidene groups and a nitrogen
heteroatom in the optimal structure for the optimal antiproliferative activity. From this SAR analysis, it
is noteworthy that we could not find (apart from a few exceptions like 6a, 7a or 6e, 8e) a very strong
relationship between the IC50 values of the two cell lines. The two cell lines correlate neither the
Pearson correlation nor the Kendall coefficient of concordance. Our results on the corresponding
calculations showed a Kendall coefficient match bigger than 1. It was 1.75, which may mean
interestingly two different ways of biological action on the two different cancer cell lines.
3.3. Physicochemical investigations
3.3.1. Experimental logP (logP):
On the basis of our former results [28, 29], we designed and performed experiments in order to obtain
experimental logP values of cyclic C5-curcuminoid compounds 6-9. The method we developed,
validated and applied is a chromatographic measurement in a reversed phase thin-layer
chromatographic (RP-TLC) setting. The theoretical base for the determination of logP by
chromatographic methods is the relation between the partition coefficient (measured with a shake-
flask) and a chromatographic retention parameter (RM for RP-TLC) based on liquid/liquid partition. RM
values (calculated using Rf) are in a linear correlation with logP values in a suitable chromatographic
system according to the
logꢀ = aꢁꢂ + b
equation. The essence of chromatographic logP determination is thus the determination of the
regression parameters of the above equation by using a set of standards (calibration set) with known
octanol/water logP values. Based on this equation the experimental logP of other compounds can be
calculated. See experimental for details.
Results from the RP-TLC measurements for experimental logP data are summarized in Table 3.
These data revealed a clear, consistent structure dependency of the measured logP values. The
model molecules are highly lipophilic, as might be expected from their chemical structures. They
contain an extended lipophilic carbon skeleton with aromatic moieties, which are compensated only a
little bit by the polarity of the carbonyl group. Polarity of the structures is under slight influence also by
the aromatic substituents.
Table 3 Experimental logP values (± sample standard deviation), determined by RP-TLC method

Compounds:
:
6
:
7
:
8
:
9
R=
: -NO
: -H
4.70 ± 0.01
5.82 ± 0.06
5.98 ± 0.07
6.40 ± 0.05
6.75 ± 0.07
5.93
3.22 ± 0.30
4.11 ± 0.17
5.10 ± 0.32
4.75 ± 0.12
7.29 ± 0.02
4.89
3.31 ± 0.03
4.14 ± 0.04
4.57 ± 0.06
5.01 ± 0.09
5.69 ± 0.15
4.54
3.62 ± 0.02
3.36 ± 0.01
3.57 ± 0.04
4.28 ± 0.01
4.04 ± 0.13
3.77
a
c
d
b
e
2
: -OCH
3
: -Cl
: -N(CH )
3 2
Average:
Apart from some exceptions like 7e or 9a, lipophilicity is decreasing in the sequence of 6, 7, 8 and 9.
This tendency is visible in general (see the average values) and in parts (see the rows in Table 3). On
the other hand, lipophilicity is growing from nitro toward dimethylamino substituent (see the columns in
Table 3) in the sequence of a, c, d, b and e along with exceptions like 7d or 9a. The exceptions
mentioned before are due to a possible self-dimerization with H-bonds of compounds 7 or 9
respectively, through their polar functional groups (NH or OH) [30, 31]. This possible dimerization
makes them less polar and so more lipophilic under the conditions of the measurement. The most
lipophilic substance is 7e with an extreme high logP value.
Our goal was also to determine if there was a correlation between the obtained logP data and the
pharmacological activity of model compounds 6-9. Therefore, we collected the experimental logP and
the corresponding IC50 values into Table 4. In order to have a more detailed insight, the data from
Table 4 underwent calculations according to Pearson correlation and Kendall coefficient of
concordance. As a result of our calculations, we may conclude, that there was no consistent linear or
logarithmic relation that could be established between antiproliferative activity and lipophilicity of our
model compounds in this study. This fact does not mean, however, that there is no positive correlation
between the nature of the structure and the biological activity at all.
Table 4 Collected experimental data from logP and IC50 measurements.

Compound
IC50 [µM]
Jurkat
logP
MCF-7
0.49
17.00
9.80
8.77
>20
4.70
6.40
5.82
5.98
6.75
3.22
4.75
4.11
5.10
7.29
3.31
5.01
4.14
4.57
5.69
3.62
4.28
3.36
3.57
4.04
0.40
6.59
4.00
2.89
>20
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
0.32
0.36
0.24
10.32
0.14
1.00
0.21
1.49
9.32
>20
0.12
>20
0.81
5.87
9.80
0.75
0.43
2.38
>20
>20
0.88
1.99
2.30
10.50
19.85
2.15
1.64
>20
8.71
11.31
>20
Median: 4.63
9.29
We can state for example that compounds with IC50 value under the median value of 2.15 ꢀM Jurkat or
9.29 ꢀM MCF-7 (which means good antiproliferative action) together with lower logP value of the
median 4.63 are promising molecules. Compounds like this in Table 4 are 7a, 7c, 8a, 8c and 9a under
Jurkat line or 7a, 7c, 8a, 8c, 9a and 9c under the MCF-7 line. The logP of these most effective
compounds are in the range of 3.22 and 4.63. There are derivatives with higher logP value, which are
practically ineffective compounds like 6e or 8e. There is only one compound (7e) in conflict with this
(Table 4). The majority of these compounds are in good accordance with Lipinski’s rule [32].
To visualize the relationship between experimental logP and IC50 data we have plotted them against
each other. The diagrams on the two cancer cell lines are in Fig 5. The most promising compounds
are in blue. They have lower lipophilicity with higher antiproliferative activity (smaller IC50 values).
Derivatives represented with red spots are more lipophilic with lower activity, except 6c, 6d and 7e on
Jurkat cells. The most promising compound is 7a, which is the most potent on both cell lines with the
lowest lipophilicity, which is also important from a pharmacokinetic point of view.

Jurkat
[CELLATART
OMÁNY]
[CELLATART
OMÁNY]
[CELLATART
OMÁNY]
10
8
[CELLATART
OMÁNY]
6
[CELLATART
4
OMÁNY]
[CELLATART
[CELLATART
[CELLATART
[CELLATART
[CELLATART
OMÁNY]
2
OMÁNY]
OMÁNY]
[CELLATART
OMÁNY]
[CELLATART
[CELLATART
OMÁNY]
[CELLATART
[CELLATART
[CELLATART
OMÁNY]
OMÁNY]
[CELLATART
OMÁNY]
0
OMÁNY]
OMÁNY]
OMÁNY]
OMÁNY]
3
4
5
6
7
8
logP
MCF-7
[CELLATAR[TCELLATART
10
OMÁNY] OMÁNY]
[CELLATART
9
8
7
6
5
4
3
2
1
0
[CELLATART
OMÁNY]
OMÁNY]
[CELLATART
OMÁNY]
[CELLATART
[CELLATART
OMÁNY]
[CELLATART
[CELLATART
OMÁNY]
OMÁNY]
[CELLATART
[CELLATART
OMÁNY]
[CELLATART
OMÁNY]
[CELLATART
OMÁNY]
OMÁNY]
3.5OMÁNY]4.5
2
2.5
3
4
5
5.5
6
6.5
7
7.5
8
logP
Fig. 5. Relationship between antiproliferative activity and lipophilicity of compounds 6-9. Experimental
logP and IC50 data of the effective derivatives are plotted against each other.
3.3.2. Predictive physicochemical calculations (ClogP, solubility in water and 3D shape):
For an evaluation of some physicochemical properties (compared to curcumin), the parameters of the
synthesized compounds were computed. Just as in our previous studies [22, 23] ChemAxon’s Marvin
Suite Plugins were used for all of the calculations [33]. Calculated logP was determined according to
three different methods of Marvin’s calculator plus the average value (Table 5).

Table 5 Computed physicochemical parameters of the investigated compounds were undertaken.
Calculated partition coefficient (ClogP) values were determined according to three methods (VG,
KLOP, PHYS) of Marvin Suite [33]. Predicted solubility at pH=7.4 of the compounds is considered to
be “Low” in this table, when it does not exceed 0.01 and is moderate if it was 0.01-0.06 mg/ml in
water.
C
P
P
log
log
Compound MW
H-bond Solubility
IC50
VG
KLOP PHYS Average (TLC)
Jurkat MCF-7
378.38 6.20
357.27 7.33
288.39 6.30
348.44 5.79
374.53 6.82
365.35 4.30
344.24 5.43
275.35 4.39
335.40 3.89
361.49 4.92
379.37 4.66
358.26 5.79
289.38 4.76
349.43 4.25
375.52 5.28
380.36 4.51
359.25 5.64
290.36 4.61
350.41 4.10
376.50 5.14
368.13 3.95
5.58
7.04
5.76
5.49
5.73
3.65
5.11
3.83
3.56
3.80
3.93
5.40
4.12
3.85
4.08
3.82
5.28
4.01
3.73
3.97
3.65
5.51
6.91
5.59
5.43
5.75
3.39
4.78
3.47
3.30
3.63
3.89
5.28
3.97
3.80
4.13
3.94
5.33
4.02
3.85
4.17
3.68
5.76
7.09
5.88
5.57
6.10
3.78
5.11
3.90
3.58
4.12
4.16
5.49
4.28
3.97
4.50
4.09
5.42
4.21
3.90
4.43
3.76
4.70
6.40
5.82
5.98
6.75
3.22
4.75
4.11
5.10
7.29
3.31
5.01
4.14
4.57
5.69
3.62
4.28
3.36
3.57
4.04
4.12
5
1
1
3
3
7
3
3
5
5
6
2
2
4
4
7
3
3
5
5
9
Low
Low
0.40
6.59
4.00
2.89
>20
0.49
17.00
9.80
8.77
>20
6a
6b
Low
6c
Low
6d
Low
6e
Low
0.32
0.36
0.24
10.32
0.14
1.00
0.21
1.49
9.32
>20
0.12
>20
7a
Low
7b
Low
0.81
5.87
9.80
0.75
0.43
2.38
>20
7c
Moderate
Moderate
Low
7d
7e
8a
Low
8b
Low
8c
Low
8d
Moderate
Low
>20
8e
0.88
1.99
2.30
10.50
19.85
0.95
1.64
>20
9a
Low
9b
Low
8.71
11.31
>20
9c
Low
9d
Moderate
Moderate
9e
9.76
Curcumin
Number of hydrogen bond forming ability of the structures, including donor or acceptor, and solubility
in water (mg/ml) were also calculated. We couldn’t find any rationale among the predicted
physicochemical data in Table 5 to explain the difference between the antiproliferative activity of
curcumin and our model compounds 6-9. There are only some slight variations in the values of the
data listed in Table 5. This can not be the cause of the great difference in pharmacological activity
between curcumin and our cyclic C5-curcuminoids. The reason for this must be somewhere else,
which leads us to investigate further in this direction.
Data in Table 5 together with our earlier findings [22, 23] however show that the software [33] we used
in our research is useful and beneficial.
Computed structures were also cleaned into 3D shape by the software used [33]. Compounds 8c and
9c as examples are available in their “ball and stick” model without their hydrogen atoms in Fig. 6.
What we can see in general is that the two benzene rings in a molecule are not in the same plane. It is

also visible, that the central ring (4-piperidone or 4-hydroxycyclohexanone respectively) is located in a
third plane. These three planes occupy a “close-to-planar” molecular shape. It is important to note that
the central rings are almost planar. Only the atom opposite to the carbonyl function is out of the plane
in an envelope-like form. Finally, the cyclic ß-dienone moiety in this cross-conjugated system keeps
the conformation of these molecules fixed.
8c
9c
Fig. 6. “Ball and stick” 3D models of compounds 8c and 9c without hydrogen atoms.
3.4. DNA binding
CD is a reliable tool for the detection of DNA binding of ligands, like drugs or any other different
molecules. The appearance of the induced circular dichroism signal (ICD) is definitive proof of the
interaction. In addition, shifts in the DNA bands are also indicative of the binding [34, 35].
There is little known about the DNA binding of cyclic C5-curcuminoids. However, we know more about
curcumin in this respect. It is known from in vitro experiments for example, that curcumin appears in
the nucleus of cultured glioma cells after incubation. It was revealed that nuclear homing is not a result
of curcumin’s DNA binding [36]. The temporal relationship of curcumin’s apoptotic induction effect and
its nuclear homing is under investigation to acquire details about the mechanism of action. This fact
among others prompted us to initiate measurements to see possible interactions between C5-
curcuminoids and DNA. For this reason, we conducted circular dichroism spectroscopic investigations
on the synthesized cyclic C5-curcuminoid derivatives in series 6-9 using natural DNA.
Curcumin, as well as 20 cyclic C5-curcuminoid derivatives 6-9 were tested on chicken erythrocyte
DNS. In the CD spectrum of curcumin, a large ICD band appeared in the ligand’s absorption region,
showing a strong interaction between the ligand molecule and the chicken erythrocyte polynucleotide
(Fig. 7). It also caused some minor shift in the DNA band, meaning the double helical structure is
slightly distorted (Fig. 7). Those derivatives containing either aliphatic or aromatic nitrogen showed
signs of interaction - a weak ICD sign appeared in the recorded spectra for example in the case of
dimethylamino substituted 6e, 7e, 8e and 9e (Fig. 7). DNA bands did not change significantly at the
same time, indicating that the polynucleotide remains in its native B-form. The binding is most
probably the result of the weak ionic interaction of the nitrogen atoms from the cyclic C5-curcuminoid
structure with the phosphate groups of DNA. In the case of other derivatives with no nitrogen atom in

their structure neither an ICD signal, nor a shift in the DNA bands was detected, indicating that no
interaction occurs between the molecules and DNA (Fig. 7).
9e
8a
Curcumin
9a
Fig. 7. Selected CD and UV titration of 0.033 mg/ml chicken erythrocyte DNA (black, lowest curve)
with 5 mM curcumin or cyclic C5-curcuminoid stock solution (final concentrations are 16.66 µg/ml, 25
µg/ml and 33.33 µg/ml respectively). Strong interaction of curcumin, very weak interaction of 9e. The
other derivatives remain intact, like for example 8a or 9a.
Although it would be consistent with an ability of these curcuminoids 6-9 to adopt a “close-to-planar”
molecular shape (Fig. 6), in contrast to curcumin, none of them showed stronger interaction with the
DNA used in this study. It would be conceivable that the very active inhibitors in Table 2 show diverse
interaction compared to the practically ineffective counterparts (Table 1). However, compounds in
these series showed similar properties under CD conditions that make it possible to generalize: based
on these data we conclude that these derivatives do not bind to DNA in vitro. Due to this finding, we
can disclose that the antiproliferative activity of these cyclic C5-curcuminoids is not due to their
interaction with DNA.

The CD spectra of compounds 6-9 can be found in the “Supplementary data” section.
4. Conclusions
The chemical susceptibility of the ß-diketone linker between the two aromatic rings in the structure of
curcumin to hydrolysis and metabolism [24, 25] has made it a crucial point to investigate structurally
modified analogs of curcumin without such shortcomings. These well-known shortcomings at the same
time are the possible reasons for the drawbacks in the bioavailability of curcumin [3]. On this basis
and as a continuation of our former SAR studies [22, 23] we have designed and synthesized twenty
cyclic C5-curcuminoids (6-9), the truncated forms of curcumin in order to have a deeper insight into the
impact of structural modifications over cytotoxic activity and/or physicochemical parameters of our
model compounds 6-9. The ß-dienone linker of these C5-curcuminoids is stable and has the same role
as the ß-diketone moiety of curcumin such as the primary pharmacophore function. We have modified
the polarity/lipophilicity of this ß-dienone linker by introducing five different substituents (nitro, chloro,
hydrogen, methoxy and dimethylamino, respectively) onto the aromatic rings dividing the group (6-9)
into five subgroups (a, b, c, d and e). The four different cyclanons (1-4 in Fig. 2) divided the group of
these model compounds further into four additional subgroups (6-9). The four subgroups have
selected moieties in the central cyclanone ring in the position opposite the carbonyl function, and
these moieties are filling the role of the secondary pharmacophore of these compounds (Fig. 4). As a
conclusion, we can state that the structural modifications in the primary and secondary
pharmacophore of these model molecules resulted in clear correlations in our SAR analysis.
Experimental and calculated data in Table 1 and 2, in relation to the two moieties A and B (Fig. 4)
suggest that there is an electron withdrawing substituent on the benzylidene groups and a nitrogen
heteroatom in the optimal structure for the optimal antiproliferative activity. The IC50 values of the
antiproliferative activity dropped to the minimum compared to curcumin, even to submicromolar in
cases close to the optimal structure. The physicochemical parameters such as molecular weight,
polarity, logP and solubility of these compounds are in good accordance with Lipinski’s rule [32]. The
most promising compound is 7a, which is the most effective (IC50 = 0.12-0.32 ꢀM), most potent (80
times of curcumin) with the lowest lipophilicity (experimental logP = 3.22) which is important also from
a pharmacokinetic point of view. There was no sign (or very weak if at all) of interaction between cyclic
C5-curcuminoids 6-9 and DNA in the CD spectra. Therefore, we can also conclude that there is no risk
for such possible and serious side effects from this source in the case of these curcuminoids. This fact
is an advantage over curcumin if we compare the results of our circular dichroism (CD) investigations.
These findings increase the knowledge about such cyclic C5-curcuminoids in order to find the optimal
structure in terms of antiproliferative activity and potential.
Appendix: Supplementary data
Details of the logP measurement method and CD spectra are provided.

Acknowledgments
HI acknowledges support from the University of Pécs, Faculty of Medicine Research Fund PTE ÁOK-
KA-34039-12/10-11. FB acknowledges support from the Hungarian Government`s Operative Program
of Economics and Innovation Support, grant number GINOP 2.3.2-15-2016-00016.
Conflicts of interests: The authors declare no conflict of interest.
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Supplementary data
Antiproliferative cyclic C5-curcuminoids without DNA binding: design, synthesis, lipophilicity
and biological activity in a SAR analysis

Imre Hubera,*, Zsuzsanna Rozmera, Zoltán Gyöngyib, Ferenc Budánb, Péter Horváthc, Eszter Kissc, Pál
Perjésia
a Department of Pharmaceutical Chemistry, University of Pécs, Pécs H-7624, Hungary
b Department of Public Health Medicine, University of Pécs, Pécs H-7624, Hungary
c Department of Pharmaceutical Chemistry, Semmelweis University, Budapest H-1092, Hungary
Content:
1. Use of RP-TLC for determination of experimental logP
2. CD (upper) and UV (below) [DrHP1] spectra of C5-curcuminoids 6-9
1. Use of RP-TLC for determination of experimental logP
Principles:
The theoretical base for the determination of logP by chromatographic methods is the relation between
the partition coefficient and a chromatographic retention parameter (RM) based on liquid/liquid
partition.
1
ꢊ
ꢁꢂ = log ꢃ − 1ꢅ = log ꢆK
ꢁꢄ
ꢋ ꢌ
ꢇꢈꢉ ꢊꢂ
where Rf is between 0 and 1, RM (calculated from Rf) can vary between +∞ to -∞, VS and VM is the
volume of the stationary and the mobile phase, respectively. VS/VM is the phase ratio, constant for the
given chromatographic system and KTLC is the chromatographic partition coefficient.
RM values (calculated using Rf) are in a linear correlation with logP values in a suitable
chromatographic system:
logꢀ = aꢁꢂ + b
The essence of chromatographic logP determination is thus the determination of the regression
parameters of the above equation by using a set of standards with known octanol/water logP values.
Based on these equations, the logP of other compounds can be calculated after TLC experiment.
Compounds of the calibration set: are cyclic chalcone derivatives.
Q-764
A-140
Q-701

logP = 2.53
logP = 4.9
logP = 2.96
TE-3
Q-506
Q-524
logP = 4.32
logP = 5.1
logP = 4.15
Compounds of the validation set:
Progesterone
Diazepam
logPSF = 3.54
logPSF = 2.82
Chalcone
Q-693
logPSF = 3.62
logPSF = 3.74
Solutions: Investigated compounds 6-9 and compounds of the calibration and validation set are
dissolved in 1:1 methanol-chloroform.
Concentration: 1 mg/ml
Standard MIX I.
Standard MIX II.
Standard MIX III.
Q-764
Q-524
A-140
Progesterone
TE-3
Q-506
Chalcone
Diazepam
Q-701
Q-693
Plate: 20 cm x 20 cm plates pre-coated with 0.25 mm layer of silanized silica gel 60F254 (Merck,
Germany; #5747)
Before using the plates are washed with methanol and dried.

Marking the plate:
•
•
•
•
The distance of the start points from the edge of the bottom layer: 1.5 cm
The distance of the first and last start point from the edge of the layer: 2.0 cm
Number of the start points: 17; distance from each other: 1.0 cm
The distance of the front from the start points: about 15.0 cm
Spotted amount:
2 µl solution
→ Standard MIX- solutions are spotted two times
→ 11 sample can be investigated on one plate
Mobile phase: methanol + water 70 : 30 (150 ml)
The paper-lined chromatographic chamber has to be saturated with the mobile phase for 30 min
before use. After development, the front is exactly marked on the plate.
Visualisation: Under UV illumination (254 nm)
The spots are marked and the distances of the center of the spots and the start points are measured.
2. CD (upper) and UV (below) [DrHP1] spectra of C5-curcuminoids 6-9
Lowest curve is for DNA. Compounds are given to DNA in three different concentrations
(upper curves).
8a
6a

9a
7a
6b
8b
9b
7b

6c
8c
7c
9c
8d
6d